In this proposed study, nanoconstructs will be engineered using recombinant technology to contain two different types of cell penetrating peptides (CPPs), i.e. amphipathic peptide, i.e., Model Amphipathic Peptide (MAP, KLALKLALKALKAALKLA), or a cationic peptide derived from HIV Tat protein (YGRKKRRQRRR). These two CPPs have previously been shown in our laboratory to have high non-specific accumulation in mammalian cells via two distinct pathways. A highly pH-sensitive histidine-glutamic acid (HE) copolymer sequence will be linked to the CPPs to prevent non-specific internalization of the construct in non-target cells, and to target internalization atthe surface of the acidic (pH 6 - 7) tumor microenvironment. According to our hypothesis, following accumulation near the acidic surface of tumor cells, the membrane activity of the CPP will be regenerated, leading to subsequent high accumulation specifically at the tumor site. This proposal will focus on two critical specific aims to evaluate HE-CPP nanoconstructs attached to two different cargo proteins, glutathione S-transferase (32 kDa) and ribonuclease A (14 kDa), (i) recombinant production and in vitro characterization of nanoconstructs in pH-sensitive binding, internalization, and penetration across multilayered cell cultures, and (ii) in vivo characterizatin of the tumor targeting and penetration. The two types of CPPs, i.e. HE-MAP and HE-TAT, will be compared in order to determine which property, amphipathicity or guanidinium charge, will lead to better pH-sensitive activation, biodistribution, and tumor penetration. Impact and Significance. The nanoconstructs described in this proposal have potential as a breakthrough technology, and can be utilized in several different areas including the exploitation of the acidic tumor microenvironment in diagnosis and targeting tumors, and in the application of CPPs in delivery of macromolecular drugs as anticancer therapeutics. Depending on whether the MAP and/or TAT-nanoconstruct displays sufficient targeting and internalization at the tumor site, these constructs will be immensely useful as carriers of macromolecular drugs to cytosolic and/or nuclear compartments of target cells. These nanoconstructs are also easily amenable to attachment of other molecules, including PEGylation to improve pharmacokinetic/biodistribution properties, targeting moieties to further enhance tumor specificity, and anti-cancer therapeutic protein or peptide domains. Therefore, the nanoconstructs have the potential to not only provide a new method in tumor targeting of drugs, but could also lead to advancement of the field of biotechnology-derived drugs (i.e. proteins and peptides) as anti-cancer therapeutics.